PROJECTSUMMARY Everybiologicalprocess,rangingfromcellmigrationtoembryogenesisandtissuemorphogenesis,reliesona cell?sabilitytoadapttochangingmechanicalenvironments.Whileweunderstandmanybiochemicalsignaling pathways involved, the mechanisms that are integrated to govern a cell?s response to mechanical forces remain a mystery. Deciphering these interactions will shed light on the mechanical changes that drive both normal and disease state processes. To reveal how the cell responds to various forces, the Robinson lab studies Dictyostelium cytokinesis, a model shape change process by which one cell divides to form two daughtercells.Thelabhasdiscoveredthatcytokinesisisdrivenbyanintegratedcontrolsystemcomposedof proteins that modulate their behavior in response to both mechanical and biochemical signals. Although we know many of the players involved in the cytokinetic control system, their biochemical interactions that allow force propagation through the cortical network are still unknown. My goal is to characterize the regulatory mechanisms that characterize these interactions, which will be critical to elucidate the mechanisms of a cell?s response to its mechanical environment. To identify the direct interactions that govern a cell?s mechanical response, we performed immunoprecipitation followed by mass spectrometry on two key nodes of the cytokineticcontrolsystem,thescaffoldingproteinIQGAP2andtheactincrosslinkercortexillinI.Thisapproach ledtothediscoveryofpotentialbindingpartnersofthesenodes.UsingacombinationofFluorescenceCross- Correlation Spectroscopy (FCCS) and Single Molecule Pulldown (SiMPull), we have discovered a potential mechanism of inhibition by a negative regulator of the system, IQGAP1. To further understand how IQGAP1 mediates inhibition, I will purify key cytoskeletal proteins and use quantitative biochemical approaches to measure binding affinities and implement a chemically-inducible dimerization system to assess the inhibitory activity of IQGAP1. In addition, I will use super-resolution imaging during both interphase and cytokinesis to characterize alterations in complexes formed by these key cytoskeletal proteinsthat allow force transduction through the network. Moreover, I will determine the cellular role of methylmalonate semialdehyde dehydrogenase (mmsdh), which catalyzes the production of propionyl-coA. Mmsdh was identified as an interactor of cortexillin I, but was also previously identified in a genetic selection in our lab. It is possible that proteins may modified by propionylation, an underappreciated post-translational modification, which may facilitate positive regulation of the cytokinetic control system. Through a combination of genetics, mass spectrometry, and biophysical analyses, I will elucidate the cellular function of mmsdh. The work proposed here will decipher the molecular mechanisms of positive and negative regulation of the contractile network. This information will be critical for understanding the cell?s ability to sense and respond to mechanical forces, yieldinginsightintobothnormaldevelopmentalprocesses,aswellasdiseasestateprogression.